Method for diagnosing an adenocarcinoma or a benign prostate pathology

ABSTRACT

The invention concerns a method for diagnosing a prostate adenocarcinoma in a male human patient without performing a prostatic biopsy, using the PSA protein (prostate specific antigen) present in the patient&#39;s blood, serum, urine or seminal fluid that involves measuring the free PSA total level, i.e., cleaved and non-cleaved; measuring the level of all or part of the cleaved free PSA; calculating the proportion of cleaved free PSA relative to the total free PSA, non-cleaved free PSA to free PSA, and/or cleaved free PSA to any of non-cleaved free, total, or complexed PSA; and diagnosing that the patient suffers from a prostate adenocarcinoma when the ratio used is not more than a reference value or a benign pathology when the ratio used is higher than the reference value.

The present application is the National Phase Application ofInternational Application No. PCT/FR99/01622, filed Jul. 5, 1999.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to a method of screening or diagnosiswhich makes it possible to detect the presence of a prostate cancer orof a benign prostate hyperplasia (BPH) in a patient, this being withoutperforming a biopsy.

2. Description of Related Art

Indeed, the prostate specific antigen, more commonly called PSA, is theprincipal marker for prostate cancer which will affect, during one'slife, one man out of six in the West. This protease of the kallikreinfamily, mainly secreted by the prostatic epithelium, is found at aconcentration of 0.5 to 5 mg/ml in the seminal fluid and at aconcentration one million times lower in the serum of a patient. Thisserum PSA level increases markedly during a prostate cancer andmoderately during benign impairments, such as BPH, acute prostatitis,and the like.

However, the area of overlap between the various pathologies isresponsible for a substantial lack of sensitivity and of specificity.Accordingly, 30 to 45%of cancers confined to the gland, whichconstitutes an early and potentially curable stage, are not detectedwith the usual threshold of 4 ng/ml whereas three patients out of fourare wrongly suspected.

In addition, it has recently been shown that in serum, PSA combined withprotease inhibitors such as α-1-antichymostrypsin (ACT), and that theuse of the free PSA to total PSA ratio made it possible to improve thespecificity of the diagnosis.

The prior art therefore shows that techniques exist which make itpossible to diagnose the development of a prostate cancer in patients.Thus, patent application WO-A-97/12245 claims a method which makes itpossible to diagnose an adenocarcinoma of the prostate (CAP) withoutbiopsy. This method consists in measuring, in the serum or in the bloodof patients, the total quantity of PSA. If this value is between 2.5 and20 ng/ml, the concentration of free PSA is also measured. The free PSAto total PSA ratio is then calculated. If this ratio is less than 7%,the diagnosis is oriented toward an adenocarcinoma of the prostate.

However, the use of a 7% threshold for the diagnosis of a cancer of theprostate is much debated by many authors, as shown by the publication byLein et al. “Relation of free PSA/total PSA in serum for differentiatingbetween patients with prostatic cancer and benign prostate hyperplasia:which cutoff should be used?”. In this document published in the journalCancer Investigation, 16(1), 45-49, 1998, it has been shown that it isdifficult to systematically differentiate a cancer or adenocarcinoma ofthe prostate from a BPH by means of this ratio.

In addition, the publication by Catalona et al., “Prostate CancerDetection in Men With Serum PSA Concentrations of 2.6 to 4.0 ng/ml andBenign Prostate Examination”, published in JAMA of 14 May 1997-Vol. 277,No. 18, demonstrates a concentration less than 4 ng/ml should be takeninto consideration in order to make an early diagnosis of a cancer ofthe prostate.

Moreover, in the seminal fluid, PSA may have internal cleavage siteswhich do not affect its three-dimensional cohesion because of theexistence of five disulfide bridges which link the various fragments ofthe molecule. The PSA thus cleaved loses its enzymatic activity and itscapacity to bind to protease inhibitors. However, in the serum, nocleaved form is generally mentioned in the prior state of the art.

No cleaved form has so far been reported in cases of benign pathologiesof the prostate and the value of the cleaved forms has not been exploredfor diagnostic purposes.

SUMMARY OF THE INVENTION

In accordance with the present invention, the diagnostic method proposedmakes it possible to compensate for the lack of efficacy of the tests inthe total PSA concentration region greater than 2 ng/ml. To this end,the molecular forms of serum PSA of patients suffering from cancer orfrom BPH have been mapped by two-dimensional electrophoresis, combinedwith chemi-luminescence detection, in order to observe all the forms ofPSA, free, complexed and cleaved.

Thus, the serum profiles of cancer sufferers are relatively homogeneous,whereas those for BPH may comprise a relatively large proportion ofcleaved forms, and of slightly more basic spots.

It is therefore established that the increase in the free PSA to totalPSA ratio in the case of BPH may be linked to the existence of cleavedPSA which is enzymatically inactive and incapable of binding to ACT, ormay be in relationship with slightly basic free PSA which may correspondto the inactive zymogenic form.

To this effect, the present invention relates to a first method ofanalysis for the purpose of the diagnosis of an adenocarcinoma of theprostate in a male human patient, without performing prostate biopsy,which uses the PSA (prostate specific antigen) protein present in ablood, serum, urine or seminal fluid sample from the patient,characterized in that it consists in:

-   measuring the level of total free PSA, that is to say cleaved or    noncleaved,-   measuring the level of all or part of the cleaved free PSA,-   calculating the proportion of cleaved free PSA relative to the total    free PSA, and-   distinguishing two cases, namely:    -   this ratio has a value less than or equal to a reference or        differentiating value, found in patients suffering from an        adenocarcinoma of the prostate, such that the patient is        suffering from an adenocarcinoma of the prostate,-   this ratio has a value greater than the reference value, found in    patients suffering from a benign pathology of the prostate, such    that the patient is suffering from a benign pathology of the    prostate, such as a benign prostate hyperplasia.

The present invention relates to a second method of analysis for thepurpose of the diagnosis of an adenocarcinoma of the prostate in a malehuman patient, without performing prostate biopsy, which uses the PSA(prostate specific antigen) protein present in a blood, serum, urine orseminal fluid sample from the patient, characterized in that it consistsin:

-   measuring the level of noncleaved free PSA,-   measuring the level of all or part of the cleaved free PSA,-   calculating the proportion of cleaved free PSA relative to the    noncleaved free PSA, and-   distinguishing two cases, namely:    -   this ratio has a value less than or equal to a reference or        differentiating value, found in patients suffering from an        adenocarcinoma of the prostate, such that the patient is        suffering from an adenocarcinoma of the prostate,    -   this ratio has a value greater than the reference value, found        in patients suffering from a benign pathology of the prostate,        such that the patient is suffering from a benign pathology of        the prostate, such as a benign prostate hyperplasia.

In these first two cases, the reference value is between 2 and 12% andpreferably between 5 and 8%.

The present invention relates to a third method of analysis for thepurpose of the diagnosis of an adenocarcinoma of the prostate in a malehuman patient, without performing prostate biopsy, which uses the PSA(prostate specific antigen) protein present in a blood, serum, urine orseminal fluid sample from the patient, characterized in that it consistsin:

-   measuring the level of total free PSA, that is to say cleaved or    noncleaved,-   measuring the level of all or part of the noncleaved free PSA,-   calculating the proportion of noncleaved free PSA relative to the    total free PSA, and-   distinguishing two cases, namely:    -   this ratio has a value greater than or equal to a reference or        differentiating value, found in patients suffering from an        adenocarcinoma of the prostate, such that the patient is        suffering from an adenocarcinoma of the prostate,    -   this ratio has a value lower than the reference value, found in        patients suffering from a benign pathology of the prostate, such        that the patient is suffering from a benign pathology of the        prostate, such as a benign prostate hyperplasia.

In this third case, the reference value is between 88 and 98% andpreferably between 93 and 95%.

The present invention relates to a fourth method of analysis for thepurpose of the diagnosis of an adenocarcinoma of the prostate in a malehuman patient, without performing prostate biopsy, which uses the PSA(prostate specific antigen) protein present in a blood, serum, urine orseminal fluid sample from the patient, characterized in that it consistsin:

-   measuring the level of total PSA, that is to say complexed and free    (cleaved or noncleaved),-   measuring the level of all or part of the cleaved free PSA,-   calculating the proportion of cleaved free PSA relative to the total    PSA, and-   distinguishing two cases, namely:    -   this ratio has a value less than or equal to a reference or        differentiating value, found in patients suffering from an        adenocarcinoma of the prostate, such that the patient is        suffering from an adenocarcinoma of the prostate,    -   this ratio has a value greater than the reference value, found        in patients suffering from a benign pathology of the prostate,        such that the patient is suffering from a benign pathology of        the prostate, such as a benign prostate hyperplasia.

In this fourth case, the reference value is between 0.1 and 2% andpreferably between 0.4 and 1%.

The present invention relates to a first method of analysis for thepurpose of the diagnosis of an adenocarcinoma of the prostate in a malehuman patient, without performing prostate biopsy, which uses the PSA(prostate specific antigen) protein present in a blood, serum, urine orseminal fluid sample from the patient, characterized in that it consistsin:

-   measuring the level of PSA complexed,-   measuring the level of all or part of the cleaved free PSA,-   calculating the proportion of cleaved free PSA relative to the    complexed PSA, and-   distinguishing two cases, namely:    -   this ratio has a value greater than or equal to a reference or        differentiating value, found in patients suffering from an        adenocarcinoma of the prostate, such that the patient is        suffering from an adenocarcinoma of the prostate,    -   this ratio has a value less than the reference value, found in        patients suffering from a benign pathology of the prostate, such        that the patient is suffering from a benign pathology of the        prostate, such as a benign prostate hyperplasia.

In this fifth case, the reference value is between 0.1 and 2.2%,preferably between 0.6 and 1.2%.

The present invention relates to a sixth method of analysis for thepurpose of the diagnosis of an adenocarcinoma of the prostate in a malehuman patient, without performing prostate biopsy, which uses the PSA(prostate specific antigen) protein present in a blood, serum, urine orseminal fluid sample from the patient, characterized in that it consistsin:

-   measuring the level of noncleaved free PSA,-   measuring the level of all or part of the cleaved free PSA,-   measuring the level of free PSA,-   measuring the level of total PSA,-   calculating the proportion of cleaved free PSA relative to the    noncleaved free PSA, called first ratio,-   calculating the proportion of free PSA relative to the total PSA,    called second ratio, and-   distinguishing four cases, namely:    -   this first ratio has a value less than or equal to a first        reference or differentiating value and this second ratio has a        value less than or equal to a second reference or        differentiating value, such that the patient is suffering from        an adenocarcinoma of the prostate,    -   this first ratio has a value greater than or equal to a first        reference or differentiating value and this second ratio has a        value greater than or equal to a second reference or        differentiating value, such that the patient is suffering from a        benign pathology of the prostate, such as a benign prostate        hyperplasia,    -   this first ratio has a value less than or equal to a first        reference or differentiating value and this second ratio has a        value greater than or equal to a second reference or        differentiating value, such that the patient has a high        probability of suffering from an adenocarcinoma of the prostate        and a low probability of suffering from a benign pathology of        the prostate, such as a benign prostate hyperplasia,    -   this first ratio has a value greater than or equal to a first        reference or differentiating value and this second ratio has a        value less than or equal to a second reference or        differentiating value, such that the patient has a high        probability of suffering from a benign pathology of the        prostate, such as a benign prostate hyperplasia, and a low        probability of suffering from an adenocarcinoma of the prostate.

In this sixth case, the first reference value is between 1 and 18%,preferably between 5 and 8% and more precisely is substantially 6.3% andthe second reference value is between 10 and 20%, preferably between 13and 17% and more precisely is substantially 15%.

Preferably, in the context of this sixth case, the high probability ofsuffering from an adenocarcinoma of the prostate is between 80 and 95%and preferably between 84 and 87% and the low probability of sufferingfrom a benign pathology of the prostate is between 5 and 20% andpreferably between 13 and 16%, whereas the high probability of sufferingfrom a benign pathology of the prostate is between 80 and 95% andpreferably between 82 and 85%, and the low probability of suffering froman adenocarcinoma of the prostate is between 5 and 20% and preferablybetween 15 and 18%.

The present invention also relates to a method of analysis for thediagnosis of an adenocarcinoma or of a benign pathology of the prostatein a patient, which uses a combination of at least two methods describedabove.

According to a variant embodiment, it is carried out when the total PSAconcentration in the blood or in the serum is greater than 2 ng/ml.

The present invention finally relates to a process using one of themethods which have just been described; it consists in:

-   detecting the presence and quantifying the free PSA in cleaved form    or in noncleaved form, and-   determining the value of the proportion of cleaved free PSA relative    to the total free PSA, relative to a reference value.

The proteins derived from the blood, serum, urine or seminal fluid fromthe patient are subjected to migration beforehand.

Preferably, the migration is carried out by two-dimensionalelectrophoresis.

The level of total PSA is assayed beforehand.

Finally and preferably, the detection is carried out bychemiluminescence after visualization by Western blotting.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures are given by way of explanatory example andhave no limiting character. They will make it possible to understand theinvention more clearly.

FIG. 1 represents a comparative table of the position of sera fromdifferent patients as a function, on the one hand, of their total PSAconcentration, which is between 2.1 and 30 ng/ml (x-axis), and, on theother hand, of their free PSA to total PSA ratio (y-axis). This table istaken from an article “Free to total Prostate-Specific Antigen (PSA)ratio is superior to total-PSA in differentiating Benign prostatehyperplasia from prostate cancer”, Vol. 156, 1964-1968, Dec. 1996,according to P. J. Van Cangh et al. The filled circles represent thecancer cases and the open circles the BPH cases.

FIG. 2 represents a two-dimensional electrophoresis, stained with silvernitrate, where the migration of PSA, in various forms, is carried out asa function, on the one hand, of the isoelectric point on the x-axis,and, on the other hand, of the molecular weight on the y-axis.

FIG. 3 represents a two-dimensional electro-phoresis of a serum from apatient suffering from a BPH, the serum containing 3.9 ng/ml of PSA.

FIG. 4 represents a two-dimensional electro-phoresis of a serum from apatient suffering from an adenocarcinoma, the serum containing 4 000ng/ml of PSA.

FIG. 5 represents a comparative table of the position of sera fromdifferent patients as a function, on the one hand, of their total PSAconcentration, which is between 1 and 34 ng/ml (x-axis) and, on theother hand, of their cleaved free PSA concentration to noncleaved freePSA concentration ratio (y-axis). The open squares represent the cancercases and the filled circles the BPH cases.

Finally, FIG. 6 represents a comparative table of the position of serafrom different patients as a function, on the one hand, of their freePSA concentration to total PSA concentration ratio (x-axis), and, on theother hand, of their cleaved free PSA concentration to noncleaved freePSA ratio (y-axis). The open squares represent the cancer cases and thefilled circles the BPH cases.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to a method for the diagnosis of prostatecancer or of benign pathology of the prostate, such as BPH, by means ofthe treatment of 100 μl in the following manner:

-   addition of 50 μl of solution in water of sodium dodecyl sulfate    (SDS) at 10% and of dithiothreitol (DTT) at 2.3%;-   heating for 5 minutes on a boiling water bath;-   dilution in QS 350 μl of solution in water of 8.3 M urea, 2 M    Thiourea, 4%    3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS),    100 mM DTT, 2% Servalyte 4-9 (Serva, Heidelberg, Germany), 0.1 g/l    Orange G;-   rehydration in a test tube under paraffin oil for 6 to 72 hours at    room temperature of a dry Immobiline strip having a nonlinear    immobilized pH gradient between pH=3 and pH=10 (Pharmacia, Uppsalla,    Sweden) with the aid of 500 μl of dilute sample;-   migration and focusing of the proteins in the sample as a function    of their isoelectric point in the Immobiline strip with the aid of    the Multiphore II system (Pharmacia) in accordance with the    supplier's recommendations at 15° C. and over 100 kV/h at more or    less 10% at 6 000 V in order to carry out a separation in the first    dimension;-   freezing of the focused Immobiline strip at −30° C. (optional);-   equilibration of the Immobiline strip for a period of 15 minutes in    a solution containing 1.5 M Tris(hydroxy-methyl)aminomethane,    hydrochloric acid pH=8.8 supplemented with 6 M urea, 2% SDS, 30%    Glycerol, 50 mg/ml Bromophenol Blue and 10 mg/ml DTT;-   equilibration of the Immobiline strip for a period of 15 minutes in    a solution containing 1.5 M Tris(hydroxy-methyl)aminomethane,    hydrochloric acid pH=8.8 supplemented with 6 M urea, 2% SDS, 30%    glycerol, 50 mg/ml Bromophenol Blue and 480 mg/ml Iodoacetamide;-   draining for 5 to 10 minutes on a moist blotting paper of the excess    equilibrating solution by depositing the Immobiline strip on the    section;-   deposition of the Immobiline strip at the top of a polyacrylamide    gel, C=2.8%, T=12% of 16×20 cm;-   covering the Immobiline strip with a 0.5% molten agarose solution in    buffer containing 25 mM Tris(hydroxymethyl)aminomethane, 192 mM    glycine, 1% SDS, at pH=8.4;-   migration as a function of their molecular weight of the proteins    previously focused in the Immobiline strip in the polyacrylamide gel    with the aid of the Protean II xi system (Bio-Rad, Hercules, United    States of America) in accordance with the supplier's recommendations    in order to carry out a separation in the second dimension;-   transfer of the proteins from the two-dimensional gel to an    Immobilon-P membrane (Millipore, Bedford, United States of America)    with the aid of the Trans-blot system (Biorad) in a solution of 10    mM 3-[cyclohexyl-amino]-1-propanesulfonic acid (CAPS). 10% methanol    at pH=11 for 16 to 24 hours at 400 mA;-   passivation for 1 hour of the binding sites which have remained free    on the membrane with the aid of the TNTM solution (15 mM    Tris(hydroxymethyl)aminomethane, 0.14 M NaCl, 0.5 ml/l Tween 20,    pH=8.0 supplemented with 5% freeze-dried skimmed milk);-   incubation for 1 hour of the membrane in a dilution containing 10    μg/ml of mouse monoclonal antibody clone 13C9E9D6G8 (Bio-Mérieux) in    TNTM solution;-   washing the membrane 3 times 10 minutes in TNTM solution;-   incubation, for 1 hour, of the membrane in a 1/5 000 dilution of    goat polyclonal antibody anti-mouse Fc coupled to horseradish    peroxidase (Jackson ImmunoResearch, West Gove, United States of    America) in TNTM solution;-   washing the membrane for twice 10 minutes in TNTM solution;-   washing the membrane for 10 minutes in a solution containing 0.1 M    Tris(hydroxymethyl)aminomethane, hydrochloric acid pH=7.6;-   visualizing with the aid of the chemiluminescent substrate of    horseradish peroxidase Super Signal Ultra (Pierce, Rockford, United    States of America) according to the supplier's recommendations;-   detection of the light emitted with the aid of the FluorS    Multi-Imager system (Bio-Rad) for 1 minute to 1 hour with a scale of    4096 levels of gray;-   quantification of the cleaved and noncleaved free PSA forms with the    aid of the MultiAnalyst software (Bio-Rad);-   calculation of the proportions of cleaved free PSA relative to the    total free PSA, or of the noncleaved free PSA relative to the total    free PSA, or of the cleaved free PSA relative to the noncleaved free    PSA, or of the cleaved PSA relative to the total PSA, or of the PSA    relative to the complexed PSA;-   determination of the diagnosis of prostate cancer according to the    reference values previously described.

The invention therefore relates to a method for the diagnosis of anadenocarcinoma of the prostate but also to a method which makes itpossible to differentiate a patient suffering from an adenocarcinoma ofthe prostate from a patient suffering from a benign pathology of theprostate, such as for example a benign prostate hyperplasia (BPH).

Thus, as is clearly represented in FIG. 1, filled circles and opencircles are represented. The filled circles represent cases of cancerswhereas the open circles relate to BPHs. It can be noted essentiallythat there is a complete blend between the cancer cases and the BPHcases. It is therefore difficult with a free PSA to total PSA ratio toreally calculate if cancer or BPH exists. An important point willhowever be noted, namely that the great majority of cancer and BPHcases, which are presented later, is above a concentration of 2.1 ng/mlof total PSA. That is the case for seventy-four of the seventy-five serafrom patients tested. Only patient 35 has a value less than 1.27 ng/ml.As a general rule, it will therefore be logical to think that below thisthreshold, the cancer or BPH cases are not very frequent. The patientshaving a total PSA concentration of less than 2 ng/ml are considered tobe healthy. The risk of their having a BPH is not quantifiable becausethe number of cases analyzed is too small to be significant.

The present invention therefore consists in differentiating cancer andBPH. To happen, two-dimensional electrophoreses are carried out asrepresented for example in FIG. 2. Three proteins or polypeptides ofdifferent sizes are observed on this electrophoresis. There is first ofall, in the top portion, a protein of substantially 34 kD, at more orless 20%, which in fact corresponds to the noncleaved PSA protein.

Below, there are two masses, namely essentially 26 kD and 21 kD, at moreor less 20%, which each correspond to a cleaved form of the PSA. Acertain proportion exists, between the cleaved and noncleaved forms ofthe free PSA, which may be used to differentiate cancer and BPH.

Thus, in the case of FIG. 3, the patient is suffering from a BPH and hisserum of course contains 2.1 ng/ml of total PSA, that is exactly 3.9 ml.When this total PSA concentration is plotted on FIG. 1, it is observedthat there is, at this level, both BPH cases and cancer cases. It isfound that when the total free PSA, that is to say cleaved ornoncleaved, is measured, that when the cleaved free PSA is alsomeasured, a cleaved free PSA to total free PSA ratio can be calculatedwhich makes it possible to make the diagnosis that the patient issuffering either from a cancer when this ratio is less than or greaterthan a reference value, or from a benign pathology of the prostate whensaid ratio is greater or less than this value.

Such values have already been described above and such a diagnosticmethod may also be applied:

-   to a ratio of the noncleaved free PSA concentration to the total    free PSA concentration, or-   to a ratio of the cleaved free PSA concentration to the noncleaved    free PSA concentration, or-   to a ratio of the cleaved free PSA concentration to the total PSA    concentration, or finally-   to a ratio of the cleaved free PSA concentration to the complexed    PSA concentration.

It is also possible to use a ratio of these ratios; that is for examplethe case in FIG. 6, with the ratio of the ratio of the free PSAconcentration to the total PSA concentration to the ratio of the cleavedfree PSA concentration to the noncleaved free PSA concentration. Thiswill be developed later.

In general, one of these methods is to be carried out when the total PSAconcentration, in the blood or in the serum, is greater than or equal to2 ng/ml.

The method therefore consists in subjecting the proteins derived fromthe blood, serum, urine or seminal fluid of the patient to migration, indetecting the presence and in quantifying the free PSA in cleaved formor in noncleaved form and in determining if the patient is healthy orsuffering from an adenocarcinoma of the prostate or from another benignpathology of the prostate according to the results obtained above.

The migration is generally carried out by two-dimensionalelectrophoresis and the detection by chemiluminescence withvisualization by Western blotting. However, it is quite possible toenvisage a technique using antibodies, which are either specific for thefree PSA in cleaved form or specific for the free PSA in noncleaved formor possibly for the bound PSA, and which does not require migrationprior to the detection of said forms.

All the results obtained are summarized in the table below:

Path- fPSA/ cPSA/ Serum ology TPSA fPSA cPSA TPSA ncPSA Patient 1 BPH3.90 0.87 0.20 22.31 30.55 Patient 2 BPH 7.50 0.83 0.09 11.07 12.11Patient 3 PCa 11.00 1.21 0.00 11.00 0.00 Patient 4 PCa 713.00 104.002.50 14.59 2.46 Patient 5 PCa 3120.00 151.00 6.22 4.84 4.30 Patient 6PCa 4000.00 567.00 21.55 14.18 3.95 Patient 7 BPH 9.13 2.84 0.04 31.111.37 Patient 8 BPH 6.22 1.15 0.13 18.49 12.60 Patient 9 PCa 7.07 1.240.03 17.54 2.61 Patient 10 BPH 4.18 2.28 0.59 31.95 35.00 Patient 11 PCa10.5 0.84 0.00 8.00 0.00 Patient 12 BPH 5.70 1.10 0.13 19.30 13.80Patient 13 BPH 9.68 2.82 0.42 29.13 17.66 Patient 14 PCa 8.09 1.00 0.1213.02 13.74 Patient 15 PCa 10.55 2.09 0.00 19.81 0.00 Patient 16 PCa5.42 0.84 0.01 15.50 0.95 Patient 17 PCa 10.00 0.69 0.04 6.90 6.30Patient 18 PCa 5.42 0.30 0.01 5.54 3.79 Patient 19 BPH 5.55 1.00 0.0418.02 4.64 Patient 20 BPH 7.58 0.72 0.08 9.50 12.01 Patient 21 BPH 4.160.97 0.06 23.32 6.56 Patient 22 BPH 4.23 0.44 0.03 10.40 7.13 Patient 23BPH 8.27 3.50 0.42 42.32 13.80 Patient 24 PCa 6.81 0.92 0.18 13.51 24.80Patient 25 PCa 29.83 1.73 0.00 5.80 0.00 Patient 26 PCa 29.98 7.15 0.0624.43 0.86 Patient 27 PCa 4.98 0.29 0.00 5.82 0.00 Patient 28 BPH 6.811.02 0.19 14.98 23.34 Patient 29 PCa 5.66 0.70 0.03 12.37 5.13 Patient30 PCa 6.77 0.61 0.00 10.00 0.00 Patient 31 PCa 7.9 1.79 0.02 22.66 0.97Patient 32 PCa 4.09 0.70 0.00 17.11 0.00 Patient 33 PCa 26.51 3.16 0.0011.92 0.00 Patient 34 PCa 5.4 0.38 0.02 7.00 4.70 Patient 35 BPH 1.270.19 0.05 14.18 31.72 Patient 36 BPH 7.7 1.19 0.18 15.45 17.36 Patient37 BPH 5.24 0.72 0.06 13.74 9.51 Patient 38 BPH 6.89 1.62 0.33 23.5125.69 Patient 39 PCa 9.58 1.23 0.00 12.84 0.36 Patient 40 BPH 5.12 0.540.04 10.55 8.62 Patient 41 PCa 7.07 1.24 0.02 17.54 1.84 Patient 42 BPH6.32 1.72 0.24 27.22 16.06 Patient 43 BPH 7.67 2.57 0.28 33.51 12.42Patient 44 PCa 12.47 0.81 0.02 6.50 2.48 Patient 45 PCa 10.57 0.70 0.006.62 0.00 Patient 46 PCa 17.49 1.09 0.01 6.23 1.26 Patient 47 PCa 10.842.56 0.00 18.29 0.00 Patient 48 PCa 7.39 1.18 0.02 14.78 2.02 Patient 49PCa 40.39 10.00 0.00 24.76 0.00 Patient 50 PCa 11.43 0.89 0.00 7.79 0.32Patient 51 PCa 46.46 3.43 0.14 7.38 4.17 Patient 52 BPH 5.03 0.46 0.059.15 11.65 Patient 53 BPH 4.01 0.52 0.10 12.97 25.11 Patient 54 PCa 5.620.20 0.00 3.56 1.11 Patient 55 PCa 9.5 0.34 0.00 3.58 1.07 Patient 56BPH 6.22 1.86 0.12 29.90 6.89 Patient 57 BPH 2.42 0.74 0.07 22.73 9.71Patient 58 BPH 5.8 1.86 0.43 32.07 29.79 Patient 59 BPH 4.52 1.05 0.0923.23 9.01 Patient 60 BPH 6.22 1.86 0.21 29.90 13.02 Patient 61 PCa 4.630.71 0.00 15.33 0.00 Patient 62 PCa 13.01 2.58 0.00 8.32 0.00 Patient 63PCa 33.96 10.00 0.00 29.45 0.00 Patient 64 PCa 58.82 6.84 0.04 11.630.56 Patient 65 PCa 20.29 3.15 0.09 11.04 2.92 Patient 66 BPH 5.01 1.170.08 23.35 7.10 Patient 67 BPH 16 2.24 0.70 14.00 45.13 Patient 68 BPH4.55 1.01 0.07 22.20 8.01 Patient 69 BPH 5.20 1.53 0.13 29.42 9.34Patient 70 BPH 7.21 1.43 0.17 19.83 13.77 Patient 71 PCa 5.56 0.86 0.0015.47 0.00 Patient 72 PCa 5.62 0.20 0.01 3.56 3.45 Patient 73 PCa 98.827.68 0.03 7.77 0.33 Patient 74 PCa 18.41 1.64 0.08 8.78 4.87 Patient 75BPH 4.29 1.48 0.22 34.50 17.71

This is a comparative study between the sera of seventy-five differentpatients.

In order to clearly understand the above table, it should be noted thatthe concentrations of total PSA (TPSA), free PSA (fPSA) and cleaved PSA(cPSA) are expressed in ng/ml. The ratios, for their part, are expressedas a percentage.

This table makes it possible to visualize rapidly the ratios which existbetween:

the, concentration of total PSA, designated hereinafter by the referenceTPSA,

the concentration of PSA bound to protease inhibitors, such as ACT,designated hereinafter by the reference bPSA,

the concentration of free PSA, designated hereinafter by the referencefPSA,

the concentration of noncleaved free PSA, designated hereinafter by thereference ncPSA, and

the concentration of cleaved free PSA, designated hereinafter by thereference cPSA.

It should be noted that the concentration of noncleaved PSA (ncPSA) isused in the right-hand column. Even if it is not given, it can be easilydeduced from the following equation:ncPSA=fPSA−cPSA

When the fPSA/TPSA ratio is taken into account, it is observed that thisratio is not at all characteristic. Thus, out of the first six patients,only patients 1 and 5 have a characteristic ratio, that is to say thatit could make it possible to distinguish a cancer case from a BPH case.

In the other cases, the ratios are between 11.00 and 14.59.Discrimination between cancer and BPH is not therefore possible. This isalso true with the other sera from patients. It is for example verydifficult to differentiate between patients 8 and 9, 12 and 15, and thelike.

Particularly striking examples are the cases of patients 22, 40 and 52who are suffering from a BPH with an fPSA/TPSA ratio always less than11, whereas over the same period, patients 26, 31, 49 and 63 aresuffering from a cancer by having a ratio which is always greater than22. Between these values of 11 and 22, there is therefore a blurred zonein respect of the diagnosis. However, numerous sera are in this zone (oreven beyond as is the case for the seven patients which have just beendescribed). The number of sera in this zone is thirty-one, that is atotal of thirty-eight sera not diagnosed with an efficient probability.This represents an error of substantially 50.67%. The use of thefPSA/TPSA ratio is in this case unsuitable for distinguishing betweenBPH and cancer.

This clearly shows the limits of this technique.

If on the other hand the ratios which exist between cPSA/bPSA,cPSA/TPSA, ncPSA/fPSA, cPSA/fPSA and cPSA/ncPSA are used, and still withreference to the first six sera from patients, it is observed that thereis a large difference between the ratios for patients 1 and 2 sufferingfrom a BPH and patients 3 to 6 suffering from a cancer.

These ratios, which use the cleaved and/or noncleaved forms of the PSA,are therefore considerably more representative of the real condition ofa patient.

Thus, in the context of the examples taken from the table, with theCPSA/TPSA ratio, in the case of a BPH, the ratio is always greater thanor equal to 1.2% and in the case of a cancer, the ratio is always lessthan 0.54%.

With the cPSA/bPSA ratio, in the case of a BPH, the ratio is alwaysgreater than 1.34% and in the case of a cancer, the ratio is always lessthan 0.63%.

With the ncPSA/fPSA ratio, in the case of a BPH, the ratio is alwaysless than 89.2% and in the case of a cancer, the ratio is always greaterthan 95.88%.

With the cPSA/fPSA ratio, in the case of a BPH, the ratio is alwaysgreater than 10.8% and in the case of a cancer, the ratio is always lessthan 4.12%.

Finally, with the cPSA/ncPSA ratio, in the case of a BPH, the ratio isalways greater than 12.11% and in the case of a cancer, the ratio isalways less than 4.3%.

The ratios, which are not representative, consist of the ratiosncPSA/TPSA, ncPSA/bPSA and fPSA/TPSA since the ratios for the threepatients 3, 4 and 6, suffering from cancer, is in the range delimited bythe ratios for patients 1 and 2, suffering from a BPH.

Because of this, it may be particularly advantageous to use acombination of different ratios, cPSA/TPSA, cPSA/bPSA, ncPSA/fPSA,cPSA/fPSA and cPSA/ncPSA, or even all these ratios, to differentiate abenign pathology of the prostate relative to a cancer of the prostate.

When patients 22, 40 and 52 are considered, it is observed that theircPSA/ncPSA ratio is 7.13 -8.62-11.65, respectively. As regards patients26, 31, 49 and 63, their cPSA/ncPSA ratio is then 0.86 -0.97-0.00-0.00,respectively.

The ratio is therefore perfectly suited to the diagnosis whichpractitioners wish carried out.

If reference is now made to FIG. 5, it will be noted that above ahorizontal straight line corresponding to 6.5% of the cPSA/ncPSA ratio,practically all the BPH cases are represented, with the exception of twocases. They are patients 7 and 19 for whom the ratios are 1.37 and 4.64,that is two cases out of thirty-three, that is to say that the error issubstantially 6%, which is considerably less than the conventionalfPSA/TPSA ratio seen above.

It will also be observed that below this reference horizontal straightline situated at 6.5% of the cPSA/ncPSA ratio, practically all thecancer cases are represented, with the exception of two cases. They arepatients 14 and 24 for whom the ratios are 13.74 and 24.80, that is twocases out of forty-two, that is to say that the error is essentially4.77%.

FIG. 6 is even more vivid. Two straight lines are observed, one verticaland the other horizontal, which intersect. The vertical straight linecorresponds to the value of 15.1% of the fPSA/TPSA ratio and thehorizontal straight line corresponds to the value of 6.2% of thecPSA/ncPSA ratio. Four cases may be deduced, they are:

-   -   this cPSA/ncPSA ratio has a value of less than or equal to 6.2%        and this fPSA/TPSA ratio has a value of less than or equal to        15.1%, such that the patient is suffering from an adenocarcinoma        of the prostate,    -   this cPSA/ncPSA ratio has a value greater than 6.2% and this        fPSA/TPSA ratio has a value greater than 15.1%, such that the        patient is suffering from a benign pathology of the prostate,        such as a benign prostate hyperplasia,    -   this cPSA/ncPSA ratio has a value of less than or equal to 6.2%        and this fPSA/TPSA ratio has a value greater than 15.1%, such        that the patient has a high probability of suffering from an        adenocarcinoma of the prostate and a low probability of        suffering from a benign pathology of the prostate, such as a        benign prostate hyperplasia,    -   this cPSA/ncPSA ratio has a value greater than 6.2%, this        fPSA/TPSA ratio has a value of less than or equal to 15.1%, such        that the patient has a high probability of suffering from a        benign pathology of the prostate, such as a benign prostate        hyperplasia, and a low probability of suffering from an        adenocarcinoma of the prostate.

Even if the reference values are given at 6.2 and 15.1%, it is possiblefor these figures to vary with another panel of sera or a differentnumber of sera. Therefore, preferably, the first reference value isbetween 1 and 18%, preferably between 5 and 8%, and the second referencevalue is between 10 and 20%, preferably between 13 and 17%.

As regards the probabilities mentioned above, in the third casedescribed above, the high probability of suffering from anadenocarcinoma of the prostate is between 84 and 87% and the lowprobability of suffering from a benign pathology of the prostate isbetween 13 and 16%. Thus, there are two cases of BPH out of fourteen intotal. However, for these third and fourth cases, these values may bebroadened because with other samples a broader range may be found, forexample 80 to 95% for the high probability, and 5 to 20% for the lowprobability.

In the fourth case, the high probability of suffering from a benignpathology of the prostate is between 82 and 85%, and the low probabilityof suffering from an adenocarcinoma of the prostate is between 15 and18%. This corresponds to two cases of cancers per twelve cases in total.

However, for these third and fourth cases, these values may be broadenedbecause with other samples, a broader range may be found, for example 80to 95% for the high probability, and 5 to 20% for the low probability.

We have available three independent variables:

-   -   A=bPSA, that is to say the PSA complexed or bound to inhibitors        (ACT and the like)    -   B=ncPSA, that is to say the noncleaved free PSA    -   C=cPSA, that is to say the cleaved free PSA        with concentrations such that A>>B>>C

Consequently, the different ratios may be written in the followingmanner and may lead to the following approximations:$\quad{{1.\quad\frac{fPSA}{TPSA}} = {\frac{B + C}{A + B + C} \approx \frac{B}{A}}}$$\quad{{2.\quad\frac{ncPSA}{TPSA}} = {\frac{B}{A + B + C} \approx \frac{B}{A}}}$$\quad{{3.\quad\frac{cPSA}{TPSA}} = {\frac{C}{A + B + C} \approx \frac{C}{A}}}$$\quad{{4.\quad\frac{ncPSA}{bPSA}} = \frac{B}{A}}$$\quad{{5.\quad\frac{cPSA}{bPSA}} = \frac{C}{A}}$$\quad{{6.\quad\frac{ncPSA}{fPSA}} = {\frac{B}{B + C} = {{1 - \frac{C}{B + C}} = {{1 - \frac{1}{1 + \frac{B}{C}}} \approx {1 - \frac{C}{B}}}}}}$$\quad{{7.\quad\frac{cPSA}{fPSA}} = {\frac{C}{B + C} = {\frac{1}{1 + \frac{B}{C}} \approx \frac{C}{B}}}}$$\quad{{8.\quad\frac{cPSA}{ncPSA}} = \frac{C}{B}}$

It therefore appears that the ratios 1, 2 and 4 lead to the -same typeof response. The ratio 1 has already been described in the prior stateof the art, which was discussed above, and leads to many falsediagnoses; consequently, the ratios 2 and 4 are not of interest.

The ratios 6, 7 and 8 are linked by a precise mathematical relationshipwhich amounts to saying that they make it possible to arrive at anidentical response.

The ratios 3 and 5 lead to an equivalent response different from thepreceding ratios.

The ratios 3 and 5 to 8 are therefore representative.

Several strategies exist for assaying cleaved or noncleaved free PSA.

Cleaved PSA has the following characteristics.

Firstly, the internal fragmentation sites do not affect thethree-dimensional cohesion of the molecule because of the existence offive disulfide bridges which link the various fragments. Secondly, afterreduction, these disulfide bridges are broken and the various PSAfragments dissociate. Thirdly, the fragmentations modify theconformation of the PSA and cause it to lose its enzymatic activity andits capacity to bind to protease inhibitors.

I—Assay of the Cleaved Forms After Reduction of the Sample:

After fragmentation or cleavage and reduction, internal PSA zones areunmasked and inaccessible epitopes on the full-length PSA are revealed.Cleaved and reduced PSA or peptides situated before and after thecleavage sites comprise such epitopes. Immunizations with these peptidesor cleaved and reduced PSA make it possible to obtain antibodiesdirected against the desired epitopes. After reduction of the sample tobe analyzed, a test using such antibodies specifically assays thecleaved PSA.

Thus, a cleaved and reduced PSA immunogen was obtained andcharacterized. Peptides situated before or after the cleavage sites weresynthesized. Immunization with cleaved and reduced PSA was carried outon mice. Ten monoclonal antibody clones specific for the epitopescharacteristic of the cleaved PSA were obtained

II—Assay of the Cleaved Forms without Reduction of the Sample:

The cleaved PSA is inactive and may comprise a conformationalmodification responsible for the loss of activity. Immunization of micewith this PSA can make it possible to obtain antibodies directed againstthese conformational epitopes. A test using these antibodies assays thecleaved PSA directly and specifically.

Predominantly cleaved inactive PSA was obtained from a supplier.Immunization was carried out on mice.

III—Assay of Noncleaved PSA After Reduction of the Sample:

After reduction of the sample, the cleaved PSA fragments whereas theamino acid chain of the noncleaved PSA remain intact. C-terminal andN-terminal peptides make it possible to carry out a detectionspecifically of the antibodies directed against the C-terminal part orthe N-terminal part or to obtain them after immunization. Afterreduction of the sample, a sandwich with an antibody directed againstthe C-terminal part and an antibody directed against the N-terminal partwould make it possible to recognize the full-length PSA.

C-terminal and N-terminal peptides of the PSA were obtained. Thesepeptides were used in order to demonstrate immunizations on mice carriedout with seminal PSA or to immunize mice directly. Antibodies weredetected in order to define pairs recognizing the noncleaved reduced PSAand not the cleaved reduced PSA.

We have available some pairs of antibodies having the expectedcharacteristics as is clearly represented by the Optical Density Valuesin the table below:

Noncleaved Monoclonal reduced PSA at Cleaved reduced antibody 100 ng/mlPSA at 100 ng/ml 4H10B4 >2 0.133 21D7C1 >2 0.103 1C7H10 >2 0.121 12E6H91.803 0.082

IV—Assay of the Noncleaved PSA Under Nonreducing Condition:

The noncleaved PSA may comprise certain conformational epitopesdifferent from the cleaved PSA (reference to section II). Peptidesoverlapping the fragmentation sites may exhibit such epitopes.Immunization on mice with these peptides or with noncleaved PSA makes itpossible to obtain antibodies directed against these epitopes. A test,using these antibodies, assays the noncleaved PSA directly andspecifically.

The peptides were synthesized. Immunization with predominantlynoncleaved PSA was carried out on mice.

V—Assay of the Enzymatically Active PSA:

The noncleaved PSA in the serum may be enzymatically active. Thus, itwas demonstrated that the cleaved PSA was enzymatically inactive but notthe opposite. An inhibitory peptide having a high affinity for activePSA may be designed by phage display and/or modification of naturalsequences. The peptide thus designed may be used to selectively capturethe enzymatically active PSA and thus allow its assay.

The protocol which follows allows the specific assay of the noncleavedPSA. It consists in carrying out the following operations:

-   -   Coating of ELISA microplate with 100 μl of antibody at 20 μg/ml        in 0.2 M Tris Maleate buffer, pH=6.2.    -   Incubation overnight at room temperature.    -   Washing with 3 times 300 μl of 0.2 M Tris Maleate buffer, 0.5%        Tween 20, pH=6.2    -   Passivation for 1 hour with 200 μl of 0.2 M Tris Maleate buffer,        3% fetal calf serum, pH=6.2.    -   Washing with 3 times 300 μl of 0.2 M Tris Maleate buffer, 0.5%        Tween 20, pH=6.2    -   Dilution of the PSA to 100 μg/ml in 0.2 M Tris Maleate buffer,        3% fetal calf serum, pH=6.2.    -   Incubation of 100 μl of PSA for 1 hour at 37° C.    -   Washing with 3 times 300 μl of 0.2 M Tris Maleate buffer, 0.5%        Tween 20, pH=6.2    -   Incubation for 1 hour at 37° C. of 100 μl of anti-biotinylated        PSA antibody 3A1B5, diluted 1/1000 in 0.2 M Tris Maleate buffer,        3% fetal calf serum, pH=6.2.    -   Washing with 3 times 300 μl of 0.2 M Tris Maleate buffer, 0.5%        Tween 20, pH=6.2    -   Incubation for 1 hour at 37° C. of 100 μl of        streptavidin-alkaline phosphatase, diluted 1/500 in 0.2 M Tris        buffer containing 0.1 M NaCl, 1 mM MgCl₂, 0.1 mm ZnCl₂, 3% fetal        calf serum, pH=6.5.    -   Washing with 3 times 300 μl of 0.2 M Tris Maleate buffer, 0.5%        Tween 20, pH=6.2    -   Visualization for 15 minutes at 37° C. with 100 μl of pNPP        substrate    -   Blocking of the reaction with 100 μl of 1 N sodium hydroxide.    -   Reading on a spectrophotometer at 405 nm with subtraction of the        background noise at 630 nm.

1. Method of analyzing PSA (prostate specific antigen) protein ratiospresent in a blood, serum, urine or seminal fluid sample of a malepatient, for the purpose of diagnosing an adenocarcinoma of the prostateor a benign prostate hyperplasia (BPH), said method comprising:measuring a level of total free PSA in the sample, wherein the totalfree PSA consists of cleaved and non-cleaved free PSA, measuring thelevel of cleaved free PSA in the sample, calculating a patient ratio ofthe cleaved free PSA level relative to the total free PSA level,comparing the patient ratio to a reference value, wherein the referencevalue is a ratio of cleaved free PSA levels relative to total free PSAlevels found in patients suffering from adenocarcinoma of the prostate,and wherein the reference value is between 2% and 12%, interpreting apatient ratio greater than the reference value as an indication that thepatient has a benign prostate hyperplasia, and interpreting a patientratio less than or equal to the reference value as an indication thatthe patient has an adenocarcinoma of the prostate.
 2. Method accordingto claim 1, wherein the reference value is between 5% and 8%.
 3. Methodaccording to claim 1, wherein the reference value is between 5% and6.2%.
 4. Method according to claim 1, wherein the reference value isapproximately 6%.
 5. Method according to claim 1, wherein said method isused in combination with at least one additional method of analyzing PSAprotein ratios present in a male patient sample.
 6. Method according toclaim 1, wherein said method is used in combination with at least oneadditional method of diagnosing a benign prostate hyperplasia oradenocarcinoma of the prostate of a male patient.
 7. Method foranalyzing PSA (prostate specific antigen) protein ratios in a blood,serum, urine or seminal fluid sample of a male patient, for the purposeof diagnosing an adenocarcinoma of the prostate, said method comprising:measuring a level of total free PSA in the sample, wherein the totalfree PSA consists of cleaved and non-cleaved PSA, measuring a level ofcleaved free PSA in the sample, calculating a patient ratio of thecleaved free PSA level relative to the total free PSA level, comparingthe patient ratio to a reference value, wherein the reference value is aratio of cleaved free PSA levels relative to total free PSA levels foundin patients suffering from adenocarcinoma of the prostate, and whereinthe reference value is between 2% and 12%, and interpreting a patientratio less than or equal to the reference value as an indication thatthe patient has an adenocarcinoma of the prostate.
 8. Method accordingto claim 7, wherein the reference value is between 5% and 8%.
 9. Methodaccording to claim 7, wherein said method is used in combination with atleast one additional method of analyzing PSA protein ratios present in amale patient sample.
 10. Method according to claim 7, wherein saidmethod is used in combination with at least one additional method ofdiagnosing an adenocarcinoma of the prostate of a male patient.